Arginine derivative wash in protein purification using affinity chromatography

ABSTRACT

The invention relates to methods for isolating a product and/or reducing turbidity and/or impurities from a load fluid comprising the product and one or more impurities by passing the load fluid through a medium, followed by at least one wash solution comprising an arginine derivative, and collecting the product using an elution solution. The invention further relates to a product prepared using a method as described herein.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Patent Application Ser. No. 60/843,084 filed Sep. 8, 2006, whichis hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This application relates to protein purification. In particular, thisapplication relates to methods for purifying a protein bound to a mediumby passing at least one wash solution containing arginine or an argininederivative through the medium, and collecting the purified protein.

BACKGROUND

With the advent of recombinant protein technology, a protein of interestcan be produced using cultured eukaryotic or prokaryotic host cell linesengineered to express the protein. The use of the desired recombinantprotein for pharmaceutical applications is generally contingent on beingable to reliably recover adequate levels of the protein from impuritiessuch as host cell proteins, protein variants, and compounds from theculture medium.

Conventional protein purification methods are designed to separate theprotein of interest from impurities based on differences in size,charge, solubility, and degree of hydrophobicity. Such methods includechromatographic methods such as affinity chromatography, ion exchangechromatography, size exclusion chromatography, hydrophobic interactionchromatography, immobilized metal affinity chromatography, andhydroxyapatite chromatography. These methods often employ a separationmedium that can be designed to selectively adhere either the protein ofinterest or the impurities. In the bind-elute mode, the desired proteinselectively binds to the separation medium and is differentially elutedfrom the medium by different solvents. In the flow-through mode, theimpurities specifically bind to the separation medium while the proteinof interest does not, thus allowing the recovery of the desired proteinin the “flow-through.”

Current methods for the purification of proteins, such as antibodies,include two or more chromatographic steps. For example, the first stepin the protein purification protocol can involve an affinitychromatography step that utilizes a specific interaction between theprotein of interest and an immobilized capture reagent. Protein Aadsorbents are particularly useful for affinity capture of proteins suchas antibodies that contain an Fc region. However, there are numerousdrawbacks to using Protein A chromatography for protein purification. Insome instances, leakage of the Protein A capture agent results incontamination of the eluted protein product, while in other instances,affinity capture does not separate protein variants, such as aggregatedforms of the protein, from the protein of interest. Additionally,varying levels of turbidity and/or precipitates can be formed in theProtein A elution pool following pH neutralization. This turbidityand/or precipitation can lead to significant product losses in theneutralized Protein A elution pool. Accordingly, there is a need forpurification methods that reduce product losses and enhance the productpurity in the elution pool.

SUMMARY OF THE INVENTION

The invention relates to methods, in part, for isolating a product froma load fluid that contains a product, such as an antibody, and one ormore impurities by passing the load fluid through a medium that bindsthe product, followed by passing at least one wash solution containingarginine or an arginine derivative through the medium, and collectingthe product using an elution solution.

In one aspect, a method for isolating a product is provided. The methodcomprises providing a load fluid comprising a product and one or moreimpurities and contacting the load fluid with a medium that can bind theproduct under conditions suitable for binding the product, therebyobtaining a bound medium. The method further comprises contacting thebound medium with one or more wash solutions comprising arginine or anarginine derivative, thereby obtaining a washed medium. The methodfurther comprises contacting the washed medium with an elution solutionunder conditions suitable for eluting the product. The eluate comprisingthe product may then be collected. The product may also be furtherpurified and/or formulated for therapeutic use.

In some embodiments of this aspect, the product is a protein, e.g., atherapeutic protein. In certain embodiments, the product is an antibody.In specific embodiments, the antibody is directed against or raised toone of the following: Growth and Differentiation Factor-8 (GDF-8),interleukin-13 (IL-13), interleukin-22 (IL-22), A-Beta, Receptor forAdvanced Glycation End products (RAGE), and 5T4. In some embodiments,the product is an antigen-binding fragment. In some embodiments theproduct is a fusion protein. In specific embodiments, the product is anIg-fusion protein. In certain embodiments, the product is an Fc-protein,an immunoconjugate, a cytokine, an interleukin, a hormone, or atherapeutic enzyme.

In some embodiments of this aspect, the medium is a matrix, a resin, ora chromatography column. In specific embodiments, the medium is aProtein A chromatography column, e.g., a recombinant Protein A column,or a Protein G chromatography column, e.g., a recombinant Protein Gcolumn.

In some embodiments of this aspect, the concentration of arginine orarginine derivative in the wash solution is about 0.1 M to about 2.0 M.In certain embodiments, the concentration of arginine or argininederivative in the wash solution is about 0.1 M to about 0.9 M. In oneembodiment, the concentration of arginine or arginine derivative in thewash solution is about 1 M. In certain other embodiments, theconcentration of arginine or arginine derivative in the wash solution isabout 1.1 M to about 2.0 M. In yet other embodiments, the concentrationof arginine or arginine derivative in the wash solution is about 0.5 Mto about 1.0 M. In yet other embodiments, the concentration of arginineor arginine derivative in the wash solution is greater than about 0.5 Mand less than about 2.0 M. In another embodiment, the concentration ofarginine or arginine derivative in the wash solution is greater thanabout 0.5 M and less than about 1.0 M. In specific embodiments, thearginine derivative is acetyl arginine, agmatine, arginic acid,N-alpha-butyroyl-L-arginine, or N-alpha-pyvaloyl arginine.

In some embodiments of this aspect, the pH of the wash solution is about4.5 to about 8.0. In certain embodiments, the pH of the wash solution isgreater than about 4.5 and less than about 8.0. In some embodiments, thepH of the wash solution is about 7.5.

In some embodiments of this aspect, the elution solution comprises oneof: sodium chloride, arginine or an arginine derivative, glycine, HEPES,and acetic acid. In certain embodiments, the elution buffer has a pH ofabout 2.0 to about 4.0. In a specific embodiment, the elution buffer hasa pH of about 3.0.

In certain embodiments of this aspect, one or more of the impurities isa host cell protein, a nucleic acid, a product variant, an endotoxin,Protein A, Protein G, a virus or a fragment thereof, a component fromthe cell culture medium, or product variant, e.g., underdisulfide-bondedproduct, low molecular weight product, high molecular weight product,truncated product and/or misfolded product. In some embodiments, whereinat least one impurity is bound to the product, the bound medium iscontacted with one or more wash solutions through the bound mediumthereby removing at least one impurity that is bound to the product.

In certain embodiments of this aspect, the eluate comprises an isolatedproduct and the purity of the isolated product is increased compared toa corresponding method in which less than about 0.1 M of arginine orarginine derivative is used in a wash solution. In some embodiments, theeluate comprises an isolated product, and the ratio of the product to atleast one impurity is increased compared to a corresponding method inwhich no detectable amount of arginine or arginine derivative is used ina wash solution. In certain embodiments, the eluate comprises a product,wherein the ratio of the product to host cell protein is increasedcompared to a corresponding method in which less than about 0.1 M ofarginine or arginine derivative is used in a wash solution. In stillfurther embodiments, the eluate comprises a product, wherein the ratioof the product to host cell protein is increased compared to acorresponding method in which no detectable amount of arginine orarginine derivative is used in a wash solution.

In another embodiment of this aspect, the turbidity of the eluate isreduced compared to a corresponding method in which less than about 0.1M of arginine or arginine derivative is used in a wash solution. In someembodiments, the turbidity of the eluate is reduced compared to acorresponding method in which no detectable amount of arginine orarginine derivative is used in a wash solution.

In another aspect, a method of isolating an antibody is provided. Themethod comprises providing a load fluid comprising the antibody and oneor more impurities, and contacting the load fluid with a Protein Amedium or a Protein G medium, wherein the medium can bind the antibodyunder conditions suitable for binding the antibody, thereby resulting ina bound medium. The method further comprises contacting the bound mediumwith one or more wash solutions, wherein at least one wash solutioncomprises arginine or an arginine derivative in a concentration greaterthan 0.5 M and less than about 1.0 M, thereby providing a washed medium.The method further comprises contacting the washed medium with anelution solution under conditions suitable for eluting the antibody; andcollecting an eluate comprising the antibody. As a result of practicingthis method, the ratio of the antibody to host cell protein in theeluate is increased and the eluate has reduced turbidity compared to aneluate recovered in a corresponding method in which no detectable amountof arginine is used in a wash solution.

In some embodiments of this aspect, the pH of the wash solution isgreater than about 5.0 and less than about 8.0.

In another aspect, a method for reducing turbidity in an eluatecomprising a product is provided. The method comprises providing a loadfluid comprising the product and one or more impurities, and contactingthe load fluid with a medium, wherein the medium can bind the productunder conditions suitable for binding the product, thereby providing abound medium. The method further comprises contacting the bound mediumwith one or more wash solutions, wherein at least one wash solutioncomprises arginine or an arginine derivative, thereby providing a washedmedium. The method further comprises contacting the washed medium withan elution solution under conditions suitable for eluting the product,thereby generating an eluate comprising the product, and neutralizingthe pH of the eluate. The method provides an eluate that has reducedturbidity compared to a corresponding method in which no detectablearginine or arginine derivative is used in a wash solution.

In some embodiments of this aspect, the pH of the neutralized eluate isbetween about 6.5 and about 8.2. In certain embodiments, the washsolution comprises arginine or an arginine derivative in a concentrationgreater than 0.5 M and less than about 1.0 M. In some embodiments, thepH of the wash solution is greater than 5.0 and less than about 8.0. Insome embodiments of this aspect, the method does not comprise anionicupstream adsorptive filtration. In certain embodiments, the product inthe eluate is further purified and/or formulated for therapeutic use.

In another aspect, a method for reducing turbidity and impurities in aneluate comprising a product is provided. The method comprises providinga load fluid comprising the product and one or more impurities, andcontacting the load fluid with a medium, wherein the medium can bind theproduct under conditions suitable for binding the product, therebyproviding a bound medium. The method further comprises contacting thebound medium with one or more wash solutions, wherein at least one washsolution comprises arginine or an arginine derivative, thereby providinga washed medium. The method further comprises contacting the washedmedium with an elution solution under conditions suitable for elutingthe product, thereby generating an eluate comprising the product, andneutralizing the. The method provides an eluate, wherein the ratio ofthe product to at least one impurity in the eluate is increased andwherein the eluate has reduced turbidity compared to a correspondingmethod in which no detectable arginine or arginine derivative is used ina wash solution.

In some embodiments of this aspect, the pH of the neutralized eluate isbetween about 6.5 and about 8.2. In certain embodiments, the washsolution comprises arginine or an arginine derivative in a concentrationgreater than 0.5 M and less than about 1.0 M. In some embodiments, thepH of the wash solution is greater than 5.0 and less than about 8.0. Insome embodiments of this aspect, the method does not comprise anionicupstream adsorptive filtration. In certain embodiments, the product inthe eluate is further purified and/or formulated for therapeutic use.The invention also relates to various methods and products as recited inthe claims appended hereto.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting the results of experiments assaying thepercent recovery (%), turbidity, HCP (“host cell proteins”) and LRV(“log removal value”) of GDF-8 mAb-1 following a Protein A column step.

FIG. 2 is a bar graph depicting the results of experiments assaying thepercent recovery (%), turbidity, HCP and LRV of GDF-8 mAb-2 following aProtein A column step.

FIG. 3 is a bar graph depicting the results of experiments assaying thepercent recovery (%), turbidity, HCP and LRV of IL-13 mAb-1 following aProtein A column step.

FIG. 4 is a bar graph depicting the results of experiments assaying thepercent recovery (%), turbidity, HCP and LRV of IL-22 mAb following aProtein A column step.

FIG. 5 is a bar graph depicting the results of experiments assaying thepercent recovery (%), turbidity, HCP and LRV of RAGE mAb following aProtein A column step.

FIG. 6 is a bar graph depicting the results of experiments assaying theturbidity IL-13 mAb-2 following a Protein A column step.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for purifying and recoveringproducts from a load fluid containing one or more impurities using aprocedure including an arginine wash or wash with an argininederivative. The invention can be applied to the large-scale preparationof proteins for therapeutic and/or diagnostic purposes.

A. DEFINITIONS

In order that the present invention may be more readily understood,certain terms as used herein are defined. Additional definitions are setforth throughout the detailed description.

The term “product” refers to a molecule produced by human or by anatural process. A “product” can include, without limitation, a protein,e.g., a therapeutic protein, including an Igfusion protein including,Fc-containing proteins. Other proteins include an immunoconjugate, acytokine, an interleukin, a hormone, a therapeutic enzyme, a virus, atherapeutic serum, a toxin, an antitoxin, a vaccine, a blood componentor derivative, or any analogous product. The protein can be a secretedprotein. The protein can be, e.g., an antibody, an antigen-bindingfragment of an antibody, a soluble receptor, a receptor fusion, acytokine, a growth factor, an enzyme, or a clotting factor. As usedherein, the terms “product” and “protein of interest” are usedinterchangeably.

The term “protein” as used herein refers to one or more polypeptidesthat can function as a unit. The term “polypeptide” as used hereinrefers to a sequential chain of amino acids linked together via peptidebonds.

A therapeutic protein can be, for example, a secreted protein.Therapeutic proteins include antibodies, antigen-binding fragments ofantibodies, soluble receptors, receptor fusions, cytokines, growthfactors, enzymes, or clotting factors, some of which are described inmore detail herein below. The above list of proteins is merely exemplaryin nature, and is not intended to be a limiting recitation. One ofordinary skill in the art will understand that any protein may be usedin accordance with the present invention and will be able to select theparticular protein to be produced based as needed. The term “conditionedculture medium” as used herein refers to the supernatant that isgenerated from the removal of cells and cellular debris by a separationmethod, such as centrifugation and/or microfiltration, from cell culturemedium that has been exposed to host cells, which may secrete desiredrecombinant polypeptide(s) of interest. Conditioned medium can contain,e.g., the secreted recombinant polypeptide, or product of interest;selected nutrients (e.g. vitamins, amino acids, cofactors, andminerals); additional growth factors/supplements including insulin; andadditional exogenous, or host cell proteins and impurities. The termconditioned culture medium includes clarified conditioned medium,filtered conditioned medium, and conditioned cell culture medium.

The term “load fluid” refers to a liquid containing the product to beisolated and one or more impurities. A load fluid contacts a medium(e.g., is passed through a medium) under the operating conditions of theinvention described below.

The term “impurity” refers to any foreign or undesirable molecule thatis present in a solution such as a load fluid. An impurity can be abiological macromolecule such as a DNA, an RNA, or a protein, other thanthe protein of interest being purified, that is also present in a sampleof the protein of interest being purified. Impurities include, forexample, undesirable protein variants, such as aggregated proteins,misfolded proteins, underdisulfide-bonded proteins, high molecularweight species, low molecular weight species and fragments, anddeamidated species; other proteins from host cells that secrete theprotein being purified, host cell DNA, components from the cell culturemedium, molecules that are part of an absorbent used for affinitychromatography that leach into a sample during prior purification steps,for example, Protein A; an endotoxin; a nucleic acid; a virus, or afragment of any of the forgoing.

The term “medium” refers to an affinity matrix or resin that can undergoa ligand-biomacromolecule interaction with a product to be isolatedduring a macromolecular separation process. The medium can be, withoutlimitation, a Protein A chromatography column or a Protein Gchromatography column.

The term “bound medium” refers to a medium bound with a product to beisolated and also bound with one or more impurities. A bound medium canbe created by passing a load fluid through a medium under conditionssuitable for binding the product.

The term “washed medium” refers to a bound medium that is washed by oneor more wash solutions, and at least one wash solution contains arginineor an arginine derivative. A washed medium can be created by contactinga bound medium with one or more wash solutions, and at least one washsolution includes arginine or an arginine derivative. In the washedmedium, the purity of the product to be isolated is generally increasedrelative to the load fluid in bound medium (i.e., the ratio of theproduct to one or more impurities is increased).

The term “bind-elute mode” refers to a product preparation technique inwhich at least one product contained in a load fluid binds to a medium(e.g., a chromatographic resin).

The term “antibody” refers to any immunoglobulin or fragment thereof,and encompasses any polypeptide comprising an antigen-binding site. Theterm includes, but is not limited to, polyclonal, monoclonal,monospecific, polyspecific, non-specific, humanized, human,single-chain, chimeric, synthetic, recombinant, hybrid, mutated,grafted, and in vitro generated antibodies. Antibody fragments includeFab, F(ab′)₂, Fv, scFv, Fd, dAb, which may retain antigen-bindingfunction. Typically, such fragments include an antigen-binding domain.

The term “IL-13” refers to interleukin-13, including full-lengthunprocessed precursor form of IL-13, as well as the mature formsresulting from post-translational cleavage. Interleukin-13 (IL-13) is apreviously characterized cytokine secreted by T lymphocytes and mastcells (McKenzie et al. (1993) Proc. Natl. Acad. Sci. USA 90:3735-39;Bost et al. (1996) Immunology 87:663-41). The term also refers to anyfragments and variants of IL-13 that maintain at least some biologicalactivities associated with mature IL-13, including sequences that havebeen modified. The term “IL-13” includes human IL-13, as well as otherIL-13 derived from vertebrate species. Several pending applicationsdisclose antibodies against human and monkey IL-13, IL-13 peptides,vectors and host cells producing the same that can be used in themethods described herein, for example, U.S. Application Publication Nos.2006/0063228A and 2006/0073148. The contents of all of thesepublications are incorporated by reference herein in their entirety.

IL-13 shares several biological activities with IL-4. For example,either IL-4 or IL-13 can cause IgE isotype switching in B cells(Tomkinson et al. (2001) J. Immunol. 166:5792-5800). Additionally,increased levels of cell surface CD23 and serum CD23 (sCD23) have beenreported in asthmatic patients (Sanchez-Guererro et al. (1994) Allergy49:587-92; DiLorenzo et al. (1999) Allergy Asthma Proc. 20:119-25). Inaddition, either IL-4 or IL-13 can upregulate the expression of MHCclass II and the low-affinity IgE receptor (CD23) on B cells andmonocytes, which results in enhanced antigen presentation and regulatedmacrophage function (Tomkinson et al., supra). These observationsindicate that IL-13 plays an important role in the development of airwayeosinophilia and airway hyperresponsiveness (AHR) (Tomkinson et al.,supra; Wills-Karp et al. (1998) Science 282:2258-61). Accordingly,inhibition of IL-13 can be useful in ameliorating the pathology ofcertain inflammatory and/or allergic conditions, including, but notlimited to, respiratory disorders, e.g., asthma; chronic obstructivepulmonary disease (COPD); other conditions involving airwayinflammation, eosinophilia, fibrosis and excess mucus production, e.g.,cystic fibrosis and pulmonary fibrosis; atopic disorders, e.g., atopicdermatitis, urticaria, eczema, allergic rhinitis; inflammatory and/orautoimmune conditions of, the skin (e.g., atopic dermatitis),gastrointestinal organs (e.g., inflammatory bowel diseases (IBD), suchas ulcerative colitis and/or Crohn's disease), liver (e.g., cirrhosis,hepatocellular carcinoma); scleroderma; tumors or cancers (e.g., softtissue or solid tumors), such as leukemia, glioblastoma, and lymphoma,e.g., Hodgkin's lymphoma; viral infections (e.g., from HTLV-1); fibrosisof other organs, e.g., fibrosis of the liver, (e.g., fibrosis caused bya hepatitis B and/or C virus).

The term “GDF-8” refers to Growth and Differentiation Factor-8 andfactors that are structurally or functionally related to GDF-8, forexample, BMP-11 and other factors belonging to the TGF-β superfamily.The term refers to the full-length unprocessed precursor form of GDF-8,as well as the mature and propeptide forms resulting frompost-translational cleavage. The term also refers to any fragments andvariants of GDF-8 that maintain at least some biological activitiesassociated with mature GDF-8, including sequences that have beenmodified. The amino acid sequences of human GDF-8, as well as GDF-8 ofother vertebrate species (including murine, baboon, bovine, and chicken)are disclosed, e.g., US 2004-0142382, US 2002-0157125, and McPherron etal. (1997) Proc. Nat. Acad. Sci. U.S.A., 94:12457-12461, the contents ofall of which are hereby incorporated by reference in their entirety.Examples of neutralizing antibodies against GDF-8 are disclosed in,e.g., US 2004-0142382, and may be used to treat or prevent conditions inwhich an increase in muscle tissue or bone density is desirable.Exemplary disease and disorders include muscle and neuromusculardisorders such as muscular dystrophy (including Duchenne's musculardystrophy); amyotrophic lateral sclerosis; muscle atrophy; organatrophy; frailty; tunnel syndrome; congestive obstructive pulmonarydisease; sarcopenia, cachexia, and other muscle wasting syndromes;adipose tissue disorders (e.g., obesity); type 2 diabetes; impairedglucose tolerance; metabolic syndromes (e.g., syndrome X); insulinresistance induced by trauma such as burns or nitrogen imbalance; andbone degenerative diseases (e.g., osteoarthritis and osteoporosis).

GDF-8, also known as myostatin, is a secreted protein and is a member ofthe transforming growth factor-beta (TGF-β) superfamily of structurallyrelated growth factors, all of which possess physiologically importantgrowth-regulatory and morphogenetic properties (Kingsley et al. (1994)Genes Dev., 8: 133-146; Hoodless et al. (1998) Curr. Topics Microbiol.Immunol., 228: 235-272). Similarly to TGF-β, human GDF-8 is synthesizedas a 375 amino acid long precursor protein. The precursor GDF-8 proteinforms a homodimer. During processing, the amino-terminal propeptide iscleaved off at Arg-266. The cleaved propeptide, known as the“latency-associated peptide” (LAP), may remain noncovalently bound tothe homodimer, thereby inactivating the complex (Miyazono et al. (1988)J. Biol. Chem. 263: 6407-6415; Wakefield et al. (1988) J. Biol. Chem.263: 7646-7654; Brown et al. (1990) Growth Factors, 3: 35-43; and Thieset al. (2001) Growth Factors, 18: 251-259). The complex of mature GDF-8with propeptide is commonly referred to as the “small latent complex”(Gentry et al. (1990) Biochemistry, 29: 6851-6857; Derynck et al. (1995)Nature, 316: 701-705; and Massague (1990) Ann. Rev. Cell Biol., 12:597-641). Other proteins are also known to bind to mature GDF-8 andinhibit its biological activity. Such inhibitory proteins includefollistatin and follistatin-related proteins (Gamer et al. (1999) Dev.Biol., 208: 222-232).

The term “RAGE” refers to the Receptor for Advanced Glycation Endproducts. RAGE is a multi-ligand cell surface member of theimmunoglobulin super-family. RAGE consists of an extracellular domain, asingle membrane-spanning domain, and a cytosolic tail. The extracellulardomain of the receptor consists of one V-type immunoglobulin domainfollowed by two C-type immunoglobulin domains. RAGE also exists in asoluble form (sRAGE). RAGE is a pattern-recognition receptor that bindsseveral different classes of endogenous molecules leading to variouscellular responses, including cytokine secretion, increased cellularoxidant stress, neurite outgrowth and cell migration. The ligands ofRAGE include advanced glycation end products (AGEs), which form inprolonged hyperglycemic states. In addition to AGEs, known ligands ofRAGE include proteins having β-sheet fibrils that are characteristic ofamyloid deposits and pro-inflammatory mediators, includingS100/calgranulins (e.g., S100A12, S100B, S100A8-A9), serum amyloid (SAA)(fibrillar form), beta-Amyloid protein (A-Beta), and high mobility groupbox-1 chromosomal protein 1 (HMGB1, also known as amphoterin). RAGE isexpressed by many cell types, e.g., endothelial and smooth muscle cells,macrophages and lymphocytes, and in many different tissues, includinglung, heart, kidney, skeletal muscle and brain. Expression is increasedin chronic inflammatory states such as rheumatoid arthritis and diabeticnephropathy. Although its physiologic function is unclear, RAGE isinvolved in the inflammatory response and may have a role in diversedevelopmental processes, including myoblast differentiation and neuraldevelopment. A number of significant human disorders are associated withan increased production of ligands for RAGE or with increased productionof RAGE itself. These disorders include, for example, many chronicinflammatory diseases, including rheumatoid and psoriatic arthritis andintestinal bowel disease, cancers, diabetes and diabetic nephropathy,amyloidoses, cardiovascular diseases and sepsis. For example, one of theligands for RAGE, HMGB-1, has been shown to be a late mediator oflethality in two models of murine sepsis, and interaction between RAGEand ligands such as HMGB1 is believed to play an important role in thepathogenesis of sepsis and other inflammatory diseases.

The term “A-Beta” refers to the principal constituent of amyloid plaqueswithin the brain. A-Beta peptide is a 4-kDa internal fragment of 39-43amino acids of a larger transmembrane glycoprotein named amyloidprecursor protein (APP). As a result of proteolytic processing of APP bydifferent secretase enzymes, A-Beta is primarily found in both a shortform, 40 amino acids in length, and a long form, ranging from 42-43amino acids in length. Part of the hydrophobic transmembrane domain ofAPP is found at the carboxy end of A-Beta, and may account for theability of A-Beta to aggregate into plaques, particularly in the case ofthe long form. Accumulation of amyloid plaques in the brain eventuallyleads to neuronal cell death. The physical symptoms associated with thistype of neural deterioration characterize Alzheimer's disease (AD). Theaccumulation of amyloid plaques within the brain is also associated withDown's syndrome and other cognitive disorders.

Several mutations within the APP protein have been correlated with thepresence of AD (see, e.g., Goate et al., Nature 349:704, 1991 (valine717to isoleucine); Chartier Harlan et al. Nature 353:844, 1991 (valine717to glycine); Murrell et al., Science 254:97, 1991 (valine717 tophenylalanine); Mullan et al., Nature Genet. 1:345, 1992 (a doublemutation changing lysine595-methionine596 to asparagine595-leucine596),each of which is incorporated herein by reference in its entirety). Suchmutations are thought to cause AD by increased or altered processing ofAPP to A-Beta, particularly processing of APP to increased amounts ofthe long form of A-Beta (i.e., A-Beta1-42 and A-Beta1-43). Mutations inother genes, such as the presenilin genes, PS1 and PS2, are thoughtindirectly to affect processing of APP to generate increased amounts oflong form A-Beta (see Hardy, TINS 20: 154, 1997, incorporated herein byreference in its entirety). In certain embodiments, anti-A-Betaantibodies are purified in accordance with the present invention.

As used herein, the term “IL-22” refers to interleukin-22, includingfull-length unprocessed precursor form of IL-22, as well as the matureforms resulting from post-translational cleavage. The term also refersto any fragments and variants of IL-22 that maintain at least somebiological activities associated with mature IL-22, including sequencesthat have been modified. The term “IL-22” includes human IL-22, as wellas other vertebrate species. The amino acid and nucleotide sequences ofhuman and rodent IL-22, as well as antibodies against IL-22 aredisclosed in, for example, U.S. Application Publication Nos.2003-0157106, 2005-0153400, 2005-0042220 and 2005-0158760, and U.S. Pat.No. 6,939,545. The contents of all of these publications areincorporated by reference herein in their entirety.

Interleukin-22 (IL-22) is a previously characterized class II cytokinethat shows sequence homology to IL-10. Its expression is up-regulated inT cells by IL-9 or Concanavalin A (ConA) (Dumoutier L. et al. (2000)Proc Natl Acad Sci USA 97(18): 10144-9). Studies have shown thatexpression of IL-22 mRNA is induced in vivo in response tolipopolysaccharide (LPS) administration, and that IL-22 modulatesparameters indicative of an acute phase response (Dumoutier L. et al.(2000) supra; Pittman D. et al. (2001) Genes and Immunity 2:172), andthat a reduction of IL-22 activity by using a neutralizing anti-IL-22antibody ameliorates inflammatory symptoms in a mouse collagen-inducedarthritis (CIA) model. Thus, IL-22 antagonists, e.g., neutralizinganti-IL-22 antibodies and fragments thereof, can be used to induceimmune suppression in vivo, for examples, for treating autoimmunedisorders (e.g., arthritic disorders such as rheumatoid arthritis);respiratory disorders (e.g., asthma, chronic obstructive pulmonarydisease (COPD)); inflammatory conditions of, e.g., the skin (e.g.,psoriasis), cardiovascular system (e.g., atherosclerosis), nervoussystem (e.g., Alzheimer's disease), kidneys (e.g., nephritis), liver(e.g., hepatitis) and pancreas (e.g., pancreatitis).

The term “host cell proteins (HCP)” refers to non-product proteinsproduced by a host cell during cell culture or fermentation.Accordingly, in some embodiments, an eluate containing a product hasHCPs present in less than 100 parts per million (ppm) HCPs (e.g., lessthan about 50 ppm, or less than about 20 ppm). HCP composition isextremely heterogeneous and dependent on the protein product andpurification procedure used. Prior to any marketing approval of abiological product for therapeutic use, the level of contaminatingproteins (such as HCPs) in the product must be quantitatively measuredaccording to the ICH and FDA guidelines.

The term “column effluent” refers to the liquid exiting a medium orcolumn during a load cycle, or in the period during which a load isbeing applied.

B. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for purifying and recoveringproducts from a load fluid containing one or more impurities using aprocedure that includes washing a bound medium with arginine or anarginine derivative. In a preferred embodiment, the medium is a ProteinA chromatography column.

In one embodiment, a product is a protein, e.g., a therapeutic protein,including peptide antibodies. In other embodiments, the product is asecreted protein; a fusion protein, e.g., a receptor fusion protein oran Ig-fusion protein, including Fc-fusion proteins; a soluble receptor;a growth factor; an enzyme; a clotting factor; an Fc-containing protein;an immunoconjugate; a cytokine; an interleukin; a hormone; or atherapeutic enzyme.

In further embodiments of the invention, the product is protein, e.g.,an antibody, that has a C_(H)2/C_(H)3 region and therefore is amenableto purification by Protein A chromatography. The term “C_(H)2/C_(H)3region” refers to those amino acid residues in the Fc region of animmunoglobulin molecule that interact with Protein A. In someembodiments, the C_(H)2/C_(H)3 region contains an intact C_(H) ² regionfollowed by an intact C_(H) ³ region. In other embodiments, theC_(H)2/C_(H)3 region contains an Fc region of an immunoglobulin.Examples of C_(H)2/C_(H)3 region-containing proteins include antibodies,immunoadhesins and fusion proteins that include a protein of interestfused to, or conjugated with, a C_(H)2/C_(H)3 region.

In certain embodiments, at least one impurity is bound to the mediumand/or the product when the load fluid is loaded to the medium, and atleast one wash solution containing arginine or an arginine derivative isused to remove the impurity that is bound to the medium and/or theproduct.

The protein can be a secreted protein. The protein can be an antibody,an antigen-binding fragment of an antibody, a soluble receptor, areceptor fusion, a cytokine, a growth factor, an enzyme, or a clottingfactor.

In some embodiments of the invention, the protein purified using themethod of the invention is an antibody or an antigen-binding fragmentthereof. As used herein, the term “antibody” includes a proteincomprising at least one, and typically two, VH domains or portionsthereof, and/or at least one, and typically two, VL domains or portionsthereof. In certain embodiments, the antibody is a tetramer of two heavyimmunoglobulin chains and two light immunoglobulin chains, wherein theheavy and light immunoglobulin chains are inter-connected by, e.g.,disulfide bonds. The antibodies, or a portion thereof, can be obtainedfrom any origin, including, but not limited to, rodent, primate (e.g.,human and non-human primate), camelid, shark as well as recombinantlyproduced, e.g., chimeric, humanized, and/or in vitro generated, e.g., bymethods well known to those of skill in the art.

Examples of binding fragments encompassed within the term“antigen-binding fragment” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAbfragment, which consists of a VH domain; (vi) a camelid or camelizedvariable domain, e.g., a VHH domain; (vii) a single chain Fv (scFv);(viii) a bispecific antibody; and (ix) one or more antigen bindingfragments of an immunoglobulin fused to an Fc region. Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv); see, e.g., Bird et al. (1988) Science242:423-26; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A.85:5879-83). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding fragment” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those skilled in the art, and the fragments are evaluated forfunction in the same manner as are intact antibodies.

In some embodiments, the term “antigen-binding fragment” encompassessingle domain antibodies. Single domain antibodies can includeantibodies whose complementary determining regions are part of a singledomain polypeptide. Examples include, but are not limited to, heavychain antibodies, antibodies naturally devoid of light chains, singledomain antibodies derived from conventional 4-chain antibodies,engineered antibodies and single domain scaffolds other than thosederived from antibodies. Single domain antibodies may be any of the art,or any future single domain antibodies. Single domain antibodies may bederived from any species including, but not limited to mouse, human,camel, llama, goat, rabbit, cow and shark. According to one aspect ofthe invention, a single domain antibody as used herein is a naturallyoccurring single domain antibody known as heavy chain antibody devoid oflight chains. Such single domain antibodies are disclosed in WO 9404678for example. For clarity reasons, this variable domain derived from aheavy chain antibody naturally devoid of light chain is known herein asa VHH or nanobody to distinguish it from the conventional VH of fourchain immunoglobulins. Such a VHH molecule can be derived fromantibodies raised in Camelidae species, for example in camel, llama,dromedary, alpaca and guanaco. Other species besides Camelidae mayproduce heavy chain antibodies naturally devoid of light chain; suchVHHs are within the scope of the invention.

An antigen-binding fragment can, optionally, further include a moietythat enhances one or more of, e.g., stability, effector cell function orcomplement fixation. For example, the antigen-binding fragment canfurther include a pegylated moiety, albumin, or a heavy and/or a lightchain constant region.

In addition, the methods of the present invention can be used to purifysmall modular immunopharmaceutical (SMIP™) drugs (TrubionPharmaceuticals, Seattle, Wash.). SMIPs are single-chain polypeptidescomposed of a binding domain for a cognate structure such as an antigen,a counterreceptor or the like, a hinge-region polypeptide having eitherone or no cysteine residues, and immunoglobulin CH2 and CH3 domains (seealso www.trubion.com). SMIPs and their uses and applications aredisclosed in, e.g., U.S. Published Patent Application. Nos.2003/0118592, 2003/0133939, 2004/0058445, 2005/0136049, 2005/0175614,2005/0180970, 2005/0186216, 2005/0202012, 2005/0202023, 2005/0202028,2005/0202534, and 2005/0238646, and related patent family membersthereof, all of which are hereby incorporated by reference herein intheir entireties.

Other than “bispecific” or “bifunctional” antibodies, an antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

In embodiments where the protein is an antibody or a fragment thereof,it can include at least one, or two full-length heavy chains, and atleast one, or two light chains. Alternatively, the antibodies orfragments thereof can include only an antigen-binding fragment (e.g., anFab, F(ab′)₂, Fv or a single chain Fv fragment). The antibody orfragment thereof can be a monoclonal or single specificity antibody. Theantibody or fragment thereof can also be a human, humanized, chimeric,CDR-grafted, or in vitro generated antibody. In yet other embodiments,the antibody has a heavy chain constant region chosen from, e.g., IgG1,IgG2, IgG3, or IgG4. In another embodiment, the antibody has a lightchain chosen from, e.g., kappa or lambda. In one embodiment, theconstant region is altered, e.g., mutated, to modify the properties ofthe antibody (e.g., to increase or decrease one or more of: Fc receptorbinding, antibody glycosylation, the number of cysteine residues,effector cell function, or complement function). Typically, the antibodyor fragment thereof specifically binds to a predetermined antigen, e.g.,an antigen associated with a disorder, e.g., a neurodegenerative,metabolic, inflammatory, autoimmune and/or a malignant disorder.Exemplary antibodies that can be separated by the methods of theinvention include, but are not limited to, antibodies against RAGE,A-Beta peptide, interleukin-13 (IL-13), interleukin-22 (IL-22), 5T4, andgrowth and differentiation factor-8 (GDF-8).

The antibody preparations used with methods described herein can be froma number of sources including, but not limited to, serum of an immunizedanimal, ascites fluid, hybridoma or myeloma supernatants, conditionedculture medium derived from culturing a recombinant cell line thatexpresses the antibody molecule, or from a cell extract ofantibody-producing cells. In one embodiment of the invention, theproduct is an antibody from conditioned culture medium of anantibody-producing recombinant cell line. Although there can be somevariation from cell line to cell line and among the various antibodyproducts, based on the disclosure herein, it is well within the purviewof one of ordinary skill in this art to adapt the invention herein to aparticular combination of antibody protein and producing cell line.

In certain embodiments, at least one impurity is bound to the mediumand/or the product when the load fluid is loaded to the medium, and atleast one wash solution containing arginine or an arginine derivative isused to remove the impurity that is bound to the medium and/or theproduct. In one embodiment of the invention, the purified productcontains less than 60% impurities (e.g., host cell proteins), in oneembodiment, 40% impurities, in one embodiment, 20% impurities, in oneembodiment, 10% impurities, in one embodiment, 5% impurities, in oneembodiment, less than 3% impurities, and in another embodiment, lessthan 1% impurities. Impurities include, but are not limited to,undesirable protein variants, such as aggregated proteins, highmolecular weight species, low molecular weight species and fragments,and deamidated species; other proteins from host cells that secrete theprotein being purified; host cell DNA; components from the cell culturemedium, molecules that are part of an absorbent used for affinitychromatography that leach into a sample during prior purification steps,for example, Protein A and Protein G; an endotoxin; a nucleic acid; avirus, or a fragment of any of the forgoing.

The medium used in a method described herein is, for example, anaffinity chromatography column, a hydrophobic interaction chromatographycolumn, an immobilized metal affinity chromatography column, a sizeexclusion chromatography column, a diafiltration, ultrafiltration, viralremoval filtration, and/or ion exchange chromatography column, a ProteinA chromatography column or a Protein G chromatography column. A ProteinA chromatography column can be, for example, PROSEP-A™ (Millipore,U.K.), Protein A Sepharose FAST FLOW™ (GE Healthcare, Piscataway, N.J.),TOYOPEARL™ 650M Protein A (TosoHass Co., Philadelphia, Pa.), orMabSelect™ column (GE Healthcare, Piscataway, N.J.).

Before contacting the medium with a load fluid, it may be necessary toadjust parameters such as pH, ionic strength, and temperature and insome instances the addition of substances of different kinds. Thus, itis an optional step to perform an equilibration of the medium by washingit with a solution (e.g., a buffer for adjusting pH, ionic strength,etc., or for the introduction of a detergent) bringing the necessarycharacteristics for binding and purification of the product.

In one embodiment of the invention, a Protein A column is equilibratedand washed with a wash solution containing arginine or an argininederivative, thereby bringing the necessary characteristics for purifyingthe product. In one embodiment of the invention, the Protein A columnmay be equilibrated using a solution containing a salt, e.g., about 100mM to about 150 mM NaPO₄, about 100 mM to about 150 mM sodium acetate,and about 100 mM to about 150 mM NaCl. The pH of the equilibrationbuffer may range from about 6.0 to about 8.0. In one embodiment, the pHof the equilibration buffer is about 7.5. The equilibration buffer maycontain about 10 mM to about 50 mM Tris. In another embodiment, thebuffer may contain about 20 mM Tris. After contacting the medium (e.g.,a Protein A column) with the load fluid, the bound medium is washed. Inaccordance with the invention, the wash solution used in the methoddescribed herein contains arginine or an arginine derivative. Thearginine derivative can be, but is not limited to, acetyl arginine,agmatine, arginic acid, N-alpha-butyroyl-L-arginine, or N-alpha-pyvaloylarginine.

The concentration of arginine or arginine derivative in the washsolution is between about 0.1 M and about 2.0 M (e.g., 0.1 M, 0.4 M, 0.5M, 1.0 M, 1.5 M, or 2.0 M), or between about 0.5 M and about 1.0 M (e.g,0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, or 1.0 M). In certain embodiments,the concentration of arginine or arginine derivative in the washsolution is about 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8M, or 0.9 M. In certain embodiments, the concentration of arginine orarginine derivative in the wash solution is about 1.1 M, 1.2 M, 1.3 M,1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2.0 M. In certainembodiments, the concentration of arginine or arginine derivative in thewash solution is greater than about 0.5 M and less than about 2.0 M(e.g., 0.55 M, 0.75 M, 1.0 M, 1.25 M, 1.5 M, or 1.75 M, or 2.0 M), orgreater than about 0.5 M and less than about 1.0 M (e.g., 0.55 M, 0.75M, or 1.0 M). In one embodiment, the concentration of arginine is not 1M. In some embodiments, the concentration of arginine or argininederivative in the wash solution is greater than 1M. In some embodiments,the concentration of arginine or arginine derivative in the washsolution is less than 1M. In further embodiments, the wash solution maycontain about 0.1 M to about 0.9 M arginine or arginine derivative. Incertain embodiments, the concentration of arginine or argininederivative can be about 0.2 M to about 0.8 M, about 0.3 M to about 0.7M, or about 0.4 M to about 0.6 M. In further embodiments, the washsolution may contain about 1.1 M to about 3.0 M, or about 1.1 M to about2.0 M arginine or arginine derivatives. In certain embodiments, theconcentration of arginine or arginine derivative is about 1.2 M to about2.8 M, about 1.3 M to about 2.6 M, about 1.4 M to about 2.4 M, about 1.5M to about 2.2 M, about 1.6 M to about 2.0 M, or about 1.8 M to about2.0 M. In certain embodiments, the concentration of arginine or argininederivative is about 1.2 M to about 1.9 M, about 1.3 M to about 1.8 M,about 1.4 M to about 1.7 M, or about 1.5 M to about 1.6 M.

The pH of the wash solution is generally between about 4.5 and about8.0, for example, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0. In samecases, the pH of the wash solution is greater than 5.0 and less thanabout 8.0, for example, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0. The washsolution may contain 20 mM to 50 mM Tris (e.g., 20 mM, 30 mM, 40 mM or50 mM). In one embodiment the bound medium is washed with 5 columnvolumes of the wash solution, followed by an elution step.

In certain embodiments of the invention, the product may be eluted froma washed medium, e.g., a Protein A column. To elute a product from aProtein A column, the washed medium is contacted with an elution buffer.In some embodiments, the elution buffer contains about 15 mM to about 50mM NaCl. In other embodiments, the elution buffer may contain about 50mM to about 150 mM arginine or arginine derivatives. In furtherembodiments, the elution buffer may contain 50 mM to 150 mM glycine. Theelution buffer may also contain about 20 mM to about 30 mM HEPES. Theelution buffer may also contain about 25 mM to about 50 mM acetic acid.The pH of the elution buffer may range from about 2.0 to about 4.0. Inone embodiment, the pH of the elution buffer is about 3.0.

The medium may optionally be cleaned, i.e., stripped and regenerated,after elution of the antibody. This procedure is typically performedregularly to minimize the building up of impurities on the surface ofthe solid phase and/or to sterilize the matrix to avoid contamination ofthe product with microorganisms.

Buffer components may be adjusted according to the knowledge of theperson of ordinary skill in the art. Sample buffer composition rangesare provided in the Examples below. Not all of the buffers or steps arenecessary, but are provided for illustration only. A high throughputscreen, as described in the Examples, may be used to efficientlyoptimize buffer conditions for Protein A column chromatography.

In one embodiment of the method, the eluate includes an isolated productand the purity of the isolated product is increased compared to acorresponding method in which less than about 0.1 M of arginine orarginine derivative is used in a wash solution.

In another embodiment, the eluate includes an isolated product, and theratio of the product to at least one impurity is increased compared to acorresponding method in which no detectable amount of arginine orarginine derivative is used in a wash solution.

In some cases, the eluate contains a product, and the ratio of theproduct to host cell protein is increased compared to a correspondingmethod in which less than about 0.1 M of arginine or arginine derivativeis used in a wash solution.

The eluate can include a product and the ratio of the product to hostcell protein is increased compared to a corresponding method in which nodetectable amount of arginine or arginine derivative is used in a washsolution.

Turbidity provides a measure of insoluble product, insoluble impurities,and insoluble complexes of product and impurities. Turbidity can also becaused by sub-cellular particulates and cell debris. In general, lowerturbidity in the eluate is associated with a more desirable quality ofthe product. Turbidity can be assayed using methods known in the art.For example, nephelometric methods (Baker et al., Trends Biotechnol.2002 April; 20(4):149-56) or optical density can be used. Opticaldensity is generally assayed by measuring absorbance at a range of about320 nm to about 650 nm.

In some cases, the turbidity of the eluate is reduced compared to theturbidity of the eluate in a corresponding method in which less thanabout 0.1 M of arginine or arginine derivative is used in a washsolution. In certain methods, the turbidity of the eluate is reducedcompared to a corresponding method in which no detectable amount ofarginine or arginine derivative is used in a wash solution.

In certain embodiments, the method of the present invention results inreduced turbidity in an eluate that contains a product. In otherembodiments, the method results in reduced turbidity as well as reducedimpurities in the eluate that contains the product compared to acorresponding method in which no detectable amount of arginine orarginine derivative is used in a wash solution. In certain embodiments,the method does not include the use of any additional upstreamfiltration, for example, an anionic upstream adsorptive filtration.

Although the purification method of the present invention can be usedalone, it may be used in combination with other purification techniques.In one embodiment, one or more processes can be used, e.g., to prepare aload fluid to reduce the load challenge of the contaminants orimpurities while employing the methods described herein. In some cases,one or more processes are used to process the eluate, e.g., to removecontaminants or impurities that are in the eluate.

The present invention also relates to a product prepared according to amethod described herein. In general, it will typically be desirable tofurther isolate and/or purify products isolated according to the presentinvention and formulate them for pharmaceutical use according tostandard methods. For proteins, see for example Protein PurificationPrinciples and Practice 2nd Edition, Springer-Verlag, New York, 1987;Higgins, S. J. and Hames, B. D. (eds.), and Deutscher, M. P., Simon, M.I., Abelson, J. N. (eds.), Guide to Protein Purification: Methods inEnzymology (Methods in Enzymology Series, Vol 182), Academic Press,1997, incorporated herein by reference. One of ordinary skill in the artwill appreciate that the exact techniques used will vary depending onthe character of the product. Products of the invention havingpharmacologic activity will be useful in the preparation ofpharmaceuticals. These may be administered to a subject or may first beformulated for delivery by any available route including, but notlimited to parenteral (e.g., intravenous), intradermal, subcutaneous,oral, nasal, bronchial, opthalmic, transdermal (topical), transmucosal,rectal, and vaginal.

A pharmaceutical composition of the product is formulated to becompatible with its intended route of administration according tomethods known in the art, see for example, Remington: The Science &Practice of Pharmacy”, 19^(th) ed., Williams & Williams, (1995), and the“Physician's Desk Reference”, 52^(nd) ed., Medical Economics, Montvale,N.J. (1998). In some embodiments, the product is formulated usingsterile water (e.g., SWFI), buffered saline (e.g., phosphate bufferedsaline), polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol), or suitable mixtures thereof.

Non-limiting examples of products that can be recovered using themethods described herein include a protein or a peptide, e.g., anantibody, an antibody fragment, a recombinant protein, a naturallysecreted protein, a protein or a peptide that is engineered to besecreted, a non-protein product that is produced by a cell, or acombination of the foregoing products.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

Experiments were conducted to identify methods that are useful forisolation of a product that is produced by a cell bacterium or tissue,in protocols that use an affinity medium as part of the isolationprocess. In these experiments, MabSelect™ Protein A column was used tobind the product.

Example 1 GDF-8 mAb-1 Comparison of Protein A Wash Buffers for HCP andTurbidity Reduction and Product Recovery

The evaluation of various wash solutions was first performed using ahigh throughput screening (HTS) method. A MabSelect™ Protein A columnwas initially loaded with conditioned culture media from a ChineseHamster Ovary (“CHO”) cell culture process. The MabSelect™ resin wasthen slurried and 100 μL of the resin slurry was dispensed into eachwell of a 96-well microtiter plate. Each well of the microtiter platewas then washed with a test solution under evaluation, and subsequentlyeluted with a low pH buffer. The elution pool from each well was assayedfor peak turbidity by A320 and for product recovery by A280. Based onthe results from the HTS experiments, test solutions that produced thehighest recovery and lowest turbidity were selected for further testingusing small-scale column scouting runs. In the column scouting runs, aMabSelect Protein A column was equilibrated with a buffer containing 10mM Tris, 100 mM NaCl pH 7.5, and loaded with CHO conditioned culturemedia containing GDF-8 mAb-1. The column was then flushed with 5 columnvolumes (CV) of the equilibration buffer, and subsequently washed with 5CVs of a test solution under evaluation. The bound product wassubsequently eluted in a low pH buffer. The neutralized peak turbiditywas measured by A320 or by a turbidimeter, product recovery wasdetermined by A280, and the HCP level was determined by an ELISA. Thecolumn sizes used for initial evaluation were 0.5 cm or 1.1 cm indiameter with bed heights from 8 to 25 cm. Table 1 summarizes the columnoperating conditions for all experiments described in Example 1.

TABLE 1 MabSelect ™ Protein A column operating conditions Phase ofVolume Linear column (Column velocity (cm Operation Solution CompositionVolumes) hr⁻¹) Equilibration 10 mM Tris, 100 mM NaCl, 5 360/450 pH 7.5Load conditioned culture NA 240 medium Post Load 10 mM Tris, 100 mMNaCl, 2 240 Flush pH = 7.5 Wash 1 Variable (see Table 2) 5 300Pre-Elution 10 mM Tris, 100 mM NaCl, 5 450 Flush pH = 7.5 Elution 50 mMNaCl, 100 mM L- 6 150 arsinine HCl, pH = 3.0 Neutrali- 2M HEPES, 5.0%(v/v) — zation pH = 8.0 addition Post Elution 50 mM Tris, pH = 8.5 4 450Flush Strip 6M guanidine HCl 2 150 Post Strip 10 mM Tris, 100 mM NaCl, 4150/450 Flush pH = 7.5 Storage 16% (v/v) ethanol 4 360

For each run, the MabSelect™ Protein A column was equilibrated with 5column volumes of 10 mM Tris, 100 mM NaCl, pH 7.5 and subsequentlyloaded to approximately 35 mg product/mL resin. The column was thenflushed with 1 column volume of equilibration buffer and 5 columnvolumes (CV) of the test wash under evaluation (see Table 2). The washphase was followed by 5 CV flush of 10 mM Tris, 100 mM NaCl, pH 7.5. Thebound product was then eluted from the column with 100 mM L-arginine, 50mM NaCl, pH 3.0. The product pool was subsequently neutralized to pH 7.5with 2 M HEPES, pH 8.0. The column was stripped with 2 CV of 6 Mguanidine HCl. The guanidine was removed from the column with 4 CV of 10mM Tris, 100 mM NaCl, pH 7.5 before being stored in 16% ethanol (4 CV).All column operations were performed at room temperature.

The various wash solutions evaluated for HCP and peak pool turbidityreduction are listed in Table 2. Severe precipitation and product losswas observed in the Protein A elution pool when the control 1M NaCl washsolution was used (FIG. 1). In addition, the HCP clearance across theProtein A column step was less than 1 log₁₀. Compared to alternate washsolutions such as 10% isopropanol (IPA), 0.5 M Guanidine-HCl (GuHCl), or2 M Tris-HCl, the arginine wash was more effective in reducing HCP andelution pool turbidity while maintaining good product recovery (FIG. 1).

TABLE 2 List of wash solutions evaluated No. Solution Composition 1 1 MNaCl, 20 mM Tris, pH 7.5 2 0.5 M arginine, 20 mM Tris, pH 7.5 3 0.5 MGuHCl, 20 mM Tris, pH 7.5 4 10% IPA, 20 mM Tris, pH 7.5 5 1 M arginine20 mM Tris, pH 7.5 6 1 M arginine, 20 mM sodium acetate, pH 5.0 7 2 MTris, pH 7.5

Example 2 GDF-8 mAb-2 Comparison of Protein A Wash Buffers for HCP andTurbidity Reduction and Product Recovery

The conditioned culture medium from a CHO cell culture processcontaining GDF-8 mAb-2 was purified at small scale using a MabSelect™Protein A column. Column sizes used for initial evaluation were 0.5 cmor 1.1 cm in diameter with bed heights from 8 cm to 25 cm. The Protein Aoperating conditions used for GDF-8 mAB-2 purification, as well as thewash solutions evaluated for HCP and peak pool turbidity reduction werethe same as those used in Example 1 (refer to Table 2 for the testsolutions evaluated).

Severe precipitation and product loss was observed in the Protein Aelution pool when the control 1M NaCl wash solution was used (FIG. 2).In addition, the HCP clearance across the Protein A column step was lessthan 1 log₁₀. Compared to alternate wash solutions such as 10%isopropanol, 0.5 M Guanidine-HCl (GuHCl), or 2 M Tris-HCl, an argininewash was more effective in removing HCP and reducing elution poolturbidity while maintaining >75% product recovery (FIG. 2).

Example 3 IL-13 mAb-1 Comparison of Protein A Wash Buffers for HCP andTurbidity Reduction and Product Recovery

The evaluation of several wash solutions for turbidity and HCP reductionacross the Protein A column step was initially performed using a highthroughput screening (HTS) method. A 25 mL column was packed withMabSelect™ resin and loaded with CHO cell conditioned culture media to afinal load challenge of 25 mg of IL-13 mAb-1 per mL of resin. The resinwas then slurried and 100 μL of the resin slurry was dispensed into eachwell of a 96-well microtiter plate. Each well of the microtiter platewas then washed with the test solution under evaluation, and the boundproduct was eluted with 50 mM glycine, 35 mM NaCl, pH 3.0. Table 3 listsall wash solutions evaluated in this study. The elution pool from eachwell was measured by A320 for peak turbidity and A280 for productrecovery.

TABLE 3 Wash solutions evaluated in HTS Screen#1 Wash Solution pHConcentration NaCl - Control 7.5 0.15-3.5 M Tween 80 with 150 mM 7.50.05-1.0% NaCl Guanidine HCl 7.5 0.1-2.0 M CTAB* 7.5 0.1-1.0% IsopropylAlcohol (IPA) 7.5 1.0-10.0% Sodium Dodecyl Sulfate 7.5 0.05-0.7% (SDS)Propylene Glycol 7.5 5.0-40.0% Propylene Glycol (low 5.0 2.0-20.0% pH)Propylene Glycol (low 6.0 5.0-30.0% pH) Tween 80 with 0.5M NaCl 7.50.05-1.0% CHAPS** 7.5 0.05-1.0% Urea 7.5 0.1-2.0 M Sodium Sulfate 7.50.1-0.8 M Sucrose 7.5 1.0-10.0% Glycine 7.5 0.1-1.0 M Glycerol 7.51.0-10.0% CaCl₂ 7.5 0.25-2.5 M CaCl₂ (low pH) 6.0 0.1-1.75 M CaCl₂ (lowpH) 5.0 0.1-1.75 M Arginine 7.5 0.1-1.0 M Arginine (low pH) 6.0 0.1-1.0M Arginine (low pH) 5.0 0.1-1.0 M Tris 7.5 1-2 M*Cetyltrimethylammoniumbromide, charged (1 mM = CMC)**3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate

The effectiveness of the wash solution under evaluation was assessed bycomparing the normalized A320 values of the neutralized elution pools.Five test solutions, Arginine, CaCl₂, guanidine HCl, IPA and Tris weremore effective than the 1M NaCl control wash at reducing neutralizedpeak pool turbidity values. Arginine, CaCl₂, Tris and IPA were furthertested using small-scale column scouting runs. A 1.1 cm (diameter)×8 cm(bed height) columns were used for these evaluations. The conditionedculture medium from CHO cell culture containing the monoclonal antibodywas purified at small scale using a MabSelect™ Protein A column operatedat room temperature. The load challenge was fixed at 30 mg/mL of resinfor these runs. The operating conditions used for the scouting runs aresummarized in Table 4. Briefly, the MabSelect™ Protein A column wasequilibrated and loaded with CHO cell conditioned culture mediumcontaining IL-13 mAb-1. The column was then washed with 5 column volumesof a high salt buffer followed by 5 column volumes of the wash solutionunder evaluation. The column was then washed with a series of low saltsolutions in preparation for the elution step. The bound product wasthen eluted with 50 mM glycine, 15 mM NaCl, pH 3.0. The neutralized peakturbidity was measured by a turbidimeter, product recovery wasdetermined by A280, and the HCP level was determined by ELISA. Theresults from this experiment are summarized in FIG. 3.

TABLE 4 Operating conditions used for scouting runs Column Volumes Flowrate Operation Solution Composition (CVs) (cm hr⁻¹) Equilibration 150 mMNaCl, 20 mM Tris; 5 300 pH = 7.5 Load conditioned culture medium — 300Post Load 1.0 M NaCl, 20 mM Tris; 5 300 Flush pH = 7.5 Wash Testsolution 5 300 Pre-Elution 35 mM NaCl, 50 mM Tris; 5 300 Flush 1 pH =7.5 Pre-Elution 5 mM NaCl, 10 mM Tris; 5 300 Flush 2 pH = 7.5 Elution 15mM NaCl, 50 mM Glycine; 5 (3CV 300 pH = 3.0 peak volume) Strip 6.0 Mguanidine HCl 5 300 Storage 16% (v/v) ethanol 5 300 Neutralization 2.0 MTris; pH 8.5 1% (v/v) 300

The neutralized peak pool turbidity values when the 1M NaCl control washsolution was used is ˜20 NTU, and is significantly lower than thatreported in Examples 1 and 2. Arginine, CaCl₂, and Tris were moreeffective at reducing neutralized peak pool turbidity compared to the 1M NaCl control. However, of the three wash solutions that were effectivefor reducing neutralized peak pool turbidity, arginine was moreeffective for reducing HCP reduction without impacting product recovery.The HCP reduction across the MabSelect™ Protein A step with 0.4 Marginine, pH 5.0 wash was 3.5-fold higher than the corresponding valueswith the 1 M NaCl control wash (FIG. 3).

Example 4 IL-22 mAb Comparison of Protein A Wash Buffers for HCP andTurbidity Reduction and Product Recovery

The evaluation of several wash solutions for turbidity and HCP reductionacross the Protein A column step was initially performed using a HTSmethod. A 25 mL column was packed with MabSelect™ resin and loaded withCHO cell conditioned media to a final load challenge of 25 mg of IL-22mAb per mL of the resin. The resin was then slurried and 100 μL of theresin slurry was dispensed into each well of a 96-well microtiter plate.Each well of the microtiter plate was then washed with the test solutionunder evaluation, and the bound product was eluted with a low pH buffer.The elution pools were then neutralized with a high pH buffer and wereassayed for peak turbidity by A320 and for product recovery by A280.Based on the results from the HTS experiments, test solutions thatproduced the highest recovery and lowest turbidity were selected forfurther testing using small-scale column scouting runs, the conditionsfor which are summarized in Table 5.

TABLE 5 Test Conditions for Column Scouting Runs Test Conditions Run 1Wash: 2 M Tris, pH 7.5 Elution: 50 mM glycine, 20 mM NaCl, pH 3.0Neutralization: 2 M HEPES, pH 8.0 Run 2 Wash: 0.5 M Arginine, 50 mMTris, pH 7.5 Elution: 50 mM glycine, 20 mM NaCl, pH 3.0 Neutralization:2 M HEPES, pH 8.0 Run 3 Wash: 2 M Tris, pH 7.5 Elution: 25 mM HEPES, 10mM NaCl, pH 3.0 Neutralization: 2 M HEPES, pH 8.0 Run 4 Wash: 2 M Tris,pH 7.5 Elution: 100 mM Arginine, 10 mM NaCl, pH 3.0 Neutralization: 2 MHEPES, pH 8.0 Run 5 Wash: 2 M Tris, pH 7.5 Elution: 50 mM glycine, 20 mMNaCl, pH 3.0 Neutralization: 2 M Tris-base

For each run, the 0.5 cm (d)×20 cm (h) MabSelect™ Protein A column wasequilibrated with 5 column volumes (CV) of 20 mM Tris, 150 mM NaCl, pH7.5 and subsequently loaded to approximately 35 mg product/mL resin. Thecolumn was then washed with 5 CV of the test solution. The wash phasewas followed by a 3 CV pre-elution flush of 5 mM Tris, 20-30 mM NaCl, pH7.5. The bound product was then eluted from the column with a low pHbuffer and neutralized to pH 7.5 with a test solution. The column wasstripped with 5 CV of 50 mM NaOH, 500 mM sodium sulfate and stored in 5CV of 16% (v/v) ethanol, 50 mM Tris, pH 7.5. All column operations wereperformed at room temperature and are summarized in Table 6.

TABLE 6 Operating Conditions for Column Scouting Runs Column VolumesFlow rate Operation Solution Composition (CVs) (cm hr⁻¹) Equilibration150 mM NaCl, 20 mM Tris; 5 300 pH 7.5 Load conditioned culture medium —300 Wash Test Solution (see Table 5) 5 300 Pre-Elution 20-30 mM NaCl, 5mM Tris; 3 300 Flush pH 7.5 Elution Test Solution (see Table 5) 5 (3CV300 peak volume) Strip 50 mM NaOH, 500 mM 5 300 sodium sulfate Storage16% (v/v) ethanol, 50 mM 5 300 Tris, pH 7.5 Neutralization Test Solution(see Table 5) 300

As shown in FIG. 4, all test conditions resulted in comparablerecoveries of product. The Arginine wash used in Run 2 and the Tris washused in Run 5 provided the greatest reduction of HCP. The Arginine washused in Run 2, however, had a nearly 2-fold lower turbidity level thanthe Tris Wash used in Run 5.

Example 5 RAGE mAb Comparison of Protein A Wash Buffers for HCP andTurbidity Reduction and Product Recovery

The evaluation of several wash solutions for turbidity and HCP reductionacross the Protein A column step was initially performed using a HTSmethod. A 25 mL column was packed with MabSelect™ resin and loaded withCHO cell conditioned culture media to a final load challenge of 25 mg ofRAGE mAb per mL of resin. The resin was then slurried and 100 μL of theresin slurry was dispensed into each well of a 96-well microtiter plate.Each well of the microtiter plate was then washed with the test solutionunder evaluation, and the bound product was eluted with a low pH buffer.The elution pools were then neutralized with a high pH buffer and wereassayed for peak turbidity by A320 and for product recovery by A280. TheHTS experiment resulted in acceptable recovery and turbidity for themajority of solutions tested. Based upon these results and priorexperience, arginine was evaluated and chosen as the wash condition forthis product.

The 5 cm (d)×23 cm (h) MabSelect™ Protein A column was equilibrated with5 column volumes (CV) of 20 mM Tris, 150 mM NaCl, pH 7.5 andsubsequently loaded to approximately 35 mg product/mL resin. The columnwas then washed with 5 CV of 0.5 M Arginine, 50 mM Tris, pH 7.5. Thewash phase was followed by a 3 CV pre-elution flush of 39 mM NaCl, 5 mMTris, pH 7.5. The bound product was then eluted from the column with 22mM NaCl, 50 mM Glycine, pH 3.0 and neutralized to pH 7.5 with 2.0 MTris, pH 8.2. The column was stripped with 5 CV of 50 mM NaOH, 500 mMsodium sulfate and stored in 5 CV of 16% (v/v) ethanol, 50 mM Tris, pH7.5. All column operations were performed at room temperature and aresummarized in Table 7.

TABLE 7 Operating Conditions for Column Scouting Runs Column VolumesFlow rate Operation Solution Composition (CVs) (cm hr⁻¹) Equilibration150 mM NaCl, 20 mM Tris; 5 300 pH 7.5 Load conditioned culture medium —300 Wash 0.5 M Arginine, 50 mM Tris, 5 300 pH 7.5 Pre-Elution 39 mMNaCl, 5 mM Tris; pH 3 300 Flush 7.5 Elution 22 mM NaCl, 50 mM 5 (3CV 300Glycine; pH 3.0 peak volume) Strip 50 mM NaOH, 500 mM 5 300 sodiumsulfate Storage 16% (v/v) ethanol, 50 mM 5 300 Tris, pH 7.5Neutralization 2.0 M Tris, pH 8.2 0.9% 300 (v/v)

As shown in FIG. 5, the arginine wash resulted in acceptable recovery ofproduct, while providing desirable levels of HCP removal and reductionin neutralized peak turbidity.

Example 6 A-Beta mAb Comparison of Protein A Wash Buffers for HCP andTurbidity Reduction and Product Recovery

The evaluation of various wash solutions was first performed using ahigh throughput screening (HTS) method. A MabSelect Protein A column wasinitially loaded with conditioned media from a Chinese Hamster Ovary(CHO) cell culture process. The MabSelect resin was then slurried and100 uL of the resin slurry was dispensed into each well of a 96-wellmicrotiter plate. Each well of the microtiter plate was then washed witha test solution under evaluation (see Table 8), and subsequently elutedwith a low pH buffer. The elution pool from each well was assayed forpeak turbidity by A320 and for product recovery by A280. Based on theresults from the HTS experiments, the test solutions that produced thehighest recovery and highest HCP removal were selected for furthertesting using small-scale column runs. These wash solutions wereArginine and CaCl₂.

TABLE 8 HTS Wash Solutions Tested Wash Excipient Concentration NaCl0.5M, 1.0M, 1.5M, 2.0M Arginine 0.5M, 1.0M, 1.5M, 2.0M CaCl₂ 0.5M, 1.0M,1.5M, 2.0M Tris 0.5M, 1.0M, 1.5M, 2.0M

In the column runs, a 1.6 cm (diameter)×15 cm (bedheight) column wasused for these evaluations. The MabSelect Protein A column wasequilibrated with a buffer containing 50 mM Tris, 0.15M NaCl pH 7.5, andloaded with CHO cell conditioned media containing A-Beta mAb to 40mg/mL. The column was then flushed with 2 column volumes (CV) of theequilibration buffer, and subsequently washed with 5 CV's of eitherArginine or CaCl₂. The column was then washed with 10 mM Tris, 10 mMNaCl pH 7.5 in preparation for the elution step. The bound product wassubsequently eluted with 50 mM glycine, 10 mM NaCl pH 3.0. Theneutralized peak turbidity was measured by A320 or by a turbidimeter,product recovery was determined by A280, and the HCP level wasdetermined by an ELISA. Table 9 summarizes the column operatingconditions for all experiments described in this Example.

TABLE 9 Protein A Column Operating Conditions Linear Velocity ColumnOperation Solution Composition Volume (CVs) (cm/hr) Equilibration 50 mMTris, 0.15M NaCl pH 7.5 5 <300 Load conditioned culture medium NA <300Post Load Flush 50 mM Tris, 0.15M NaCl pH 7.5 2 <300 Wash 1: 50 mM TrispH 7.5 0.5M Arginine 5 <300 w/ 1.0M Arginine 0.5M CaCl₂ 1.0M CaCl₂ Wash2 10 mM Tris, 10 mM NaCl pH 7.5 5 <300 Elution 50 mM glycine, 10 mM NaClpH 3.0 4 <300 Neutralization 2M Hepes pH 8.5 or 2M Tris pH 9.0 ~1% <300Strip 50 mM NaOH, 0.5M sodium sulfate 5 <300 Strip Wash 50 mM Tris,0.15M NaCl pH 7.5 5 <300 Storage 16% (v/v) ethanol <300

TABLE 10 Protein A Data for Wash Studies Wash Conditions % Recovery byA280 HCP ppm 0.5 M Arginine 87 9300 0.5 M Arginine 87 12,700 0.5 M CaCl286 9000 0.5 M CaCl2 80 10,600 1.0 M CaCl2 80 11,500

Table 10 shows the results for recovery and HCP values for the washexperiments for A-Beta mAb. Arginine and CaCl2 were comparable washsolutions for reduction of host cell protein and final peak poolturbidity. 0.5M Arginine was chosen as the wash solution for the A-BetamAb process based the lower loss of product during the wash.

Example 7 IL-13 mAb-2 Comparison of Protein A Wash Buffers for HCP andTurbidity Reduction and Product Recovery

The evaluation of several wash solutions for turbidity reduction acrossthe Protein A column step was initially performed using a highthroughput screening (HTS) method. A 25 mL column was packed withMabSelect™ resin and loaded with CHO conditioned media to a final loadchallenge of 50 mg of IL-13 mAb-2 per mL of resin. The resin was thenslurried and 100 uL of the resin slurry was dispensed into each well ofa 96-well microtiter plate. Each well of the microtiter plate was thenwashed with a test solution under evaluation, the bound product waseluted, and the acidic elution pool neutralized.

For the HTS, various excipient washes, elution buffers and titrants,were utilized combinatorially and with varying concentrations. Excipientwashes utilized in the HTS were calcium chloride, sodium chloride, Tris,and arginine. Elution buffers utilized in the HTS were glycine, HEPES,and acetic acid together with varying concentrations of NaCl. Titrantsutilized in the HTS were Tris, HEPES and Imidazole.

Using A320 as a surrogate for precipitation, the effectiveness of thewash solutions was assessed by comparing the normalized A320 values ofthe neutralized elution pools. Arginine proved most effective atreducing the A320 readings of the eluted peak. Based on the results fromthe HTS experiments, test solutions that produced the highest recoveryand lowest turbidity were selected for further testing using small-scalecolumn scouting runs. This lead to the further testing of calciumchloride, arginine, and sodium chloride (control) as wash buffers insmall-scale column trials.

The conditioned culture medium from a CHO culture process containingIL-13 mAb-2 was purified at small scale using a MabSelect™ Protein Acolumn operated at room temperature. Column sizes used for the initialevaluation were 1.1 cm in diameter with bed heights from 20 cm to 25 cm.The load challenge was fixed at 35 mg/mL of resin for these runs. Theconditions used for the scouting runs are summarized in Table 11.

TABLE 11 Operating conditions used for scouting runs Column Volumes Flowrate Operation Solution Composition (CVs) (cm hr − 1) Equili- 150 mMNaCl, 50 mM Tris; 5 300 bration pH = 7.5 Load conditioned culture medium— 240 Post Load 150 mM NaCl, 50 mM Tris; 5 300 Flush pH = 7.5 Wash Testsolution (see Table 12) 5 300 Pre- 10 mM Tris, NaCl concentration 3 300Elution equal to elution buffer, Flush pH = 7.5 Elution See Table 13 6CV 300 (3 CV peak volume) Strip 50 mM NaOH, 500 mM sodium 5 180 sulfateStorage 16% (v/v) ethanol, 50 mM Tris, 5 300 150 mM NaCl, pH = 7.5Neutral- See Table 13 ~1-5% ization (v/v)

TABLE 12 List of wash solutions evaluated Reference Solution Composition1 0.5 M arginine, 20 mM Tris, pH 7.5 2 0.5 M calcium chloride, 20 mMTris, pH 7.5 3 0.5 M sodium chloride, 20 mM Tris, pH 7.5 4 2.0 Marginine, 20 mM Tris, pH 7.5 5 1.5 M calcium chloride, 20 mM Tris, pH7.5 6 1.0 M arginine, 20 mM Tris, pH 7.5 7 1.0 M calcium chloride, 20 mMTris, pH 7.5 7 2.0 M arginine, 20 mM Tris, pH 7.5

TABLE 13 List of elution and titration buffers employed ReferenceElution Buffer A 50 mM glycine, 20 mM NaCl, pH 3.1 B 50 mM glycine, 50mM NaCl, pH 3.1 C 50 mM acetic acid, 20 mM NaCl, pH 3.1 Titrant D 2.0 MTris, pH 9.0 E 2.0 M HEPES, pH 9.0 F 1.0 M Imidazole, pH 8.0

Briefly, the MabSelect™ Protein A column was equilibrated and loadedwith CHO conditioned medium containing IL-13 mAb-2. The column was thenwashed with 5 CVs of a moderate salt buffer followed by 5 CVs of thewash solution under evaluation. The column was then washed with 3 CVs ofa low salt solution in preparation for the elution step. The boundproduct was then eluted with solution containing either glycine oracetic acid as the buffering species with a pH of 3.1. This peak poolwas then independently neutralized with three different titrants. Theneutralized peak turbidity was measured with a turbidimeter, productrecovery was measured by A280, and the HCP level was quantified withELISA. The results from this experiment are summarized in FIG. 6.

At every concentration trialed and with various elution buffers (seeTable 13), arginine was found to be more effective than calcium chlorideor sodium chloride at reducing acidic and neutralized peak poolturbidity. This finding was consistent whether the acidic pools wereneutralized with Tris, HEPES, or imidazole titrant (see Table 13).

In addition, it was demonstrated that the effect of an arginine wash wasnot restricted to a final, discrete pH of the neutralized pool. Across apH range of 7.5-8.2 (e.g., 7.5, 7.7, 7.9, 8.0, 8.1, and 8.2), theneutralized peak turbidity values decrease as the pH increases. Acrossthis same range, however, the employment of arginine wash alwaysresulted in a lower NTU than if calcium chloride was used.

The recovery of loaded product was >95% for each excipient wash trialed.HTS data indicated that measurably less recovery would be obtained bywashing with >1.5 M calcium chloride, which is why this concentrationwas not trialed.

The examined wash species could not be differentiated based on the finalconcentration of HCP, HMW, and Protein A in the peak pool. However, ananalysis of the wash fraction eluted with a 1.0 M arginine washconsistently revealed significant levels of HCP. HCP levels in the washfraction were ˜49000 ppm while only ˜22000 ppm in the peak. This isevidence that the arginine wash selectively removes HCP.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

1. A method for isolating a product, the method comprising: (a)providing a load fluid comprising a product and one or more impurities,wherein the product is an Fc-containing monoclonal antibody; (b)contacting the load fluid with a medium, wherein the medium is a ProteinA chromatography column, and wherein the medium can bind the productunder conditions suitable for binding the product, thereby providing abound medium; (c) contacting the bound medium with one or more washsolutions, wherein at least one wash solution comprises an argininederivative, thereby providing a washed medium, wherein the argininederivative is acetyl arginine, agmatine, arginic acid,N-alpha-butyroyl-L-arginine or N-alpha-pyvaloyl arginine; (d) contactingthe washed medium with an elution solution under conditions suitable foreluting the product, and (e) collecting an eluate comprising theproduct.
 2. The method of claim 1, wherein the monoclonal antibody isspecific for GDF-8.
 3. The method of claim 1, wherein the monoclonalantibody is specific for IL-13.
 4. The method of claim 1, wherein themonoclonal antibody is specific for IL-22.
 5. The method of claim 1,wherein the monoclonal antibody is specific for RAGE.
 6. The method ofclaim 1, wherein the monoclonal antibody is specific for A-Beta.
 7. Themethod of claim 1, wherein the concentration of arginine derivative inthe wash solution is about 0.1 M to about 2.0 M.
 8. The method of claim1, wherein the concentration of arginine derivative in the wash solutionis about 0.1 M to about 0.9 M.
 9. The method of claim 1, wherein theconcentration of arginine derivative in the wash solution is about 1.1 Mto about 2.0 M.
 10. The method of claim 1, wherein the concentration ofarginine derivative in the wash solution is about 0.5 M.
 11. The methodof claim 1, wherein the pH of the wash solution is about 4.5 to about8.0.
 12. The method of claim 1, wherein the pH of the wash solution isgreater than 4.5 and less than about 8.0.
 13. The method of claim 1,wherein the pH of the wash solution is about 7.5.
 14. The method ofclaim 1, wherein one or more of the impurities are a host cell protein,a nucleic acid, a product variant, or an endotoxin.
 15. The method ofclaim 1, wherein one or more of the impurities is a virus or fragmentthereof.
 16. The method of claim 1, wherein the elution solutioncomprises at least one of sodium chloride, arginine or an argininederivative, glycine, HEPES, and acetic acid, wherein the argininederivative is acetyl arginine, agmatine, arginic acid,N-alpha-butyroyl-L-arginine, or N-alpha-pyvaloyl arg mine.
 17. Themethod of claim 1, wherein the elution solution has a pH of betweenabout 2 and about
 4. 18. The method of claim 1, wherein the eluatecomprises an isolated product and the purity of the isolated product isincreased compared to a corresponding method in which less than about0.1 M of arginine derivative is used in a wash solution.
 19. The methodof claim 1, wherein the eluate comprises an isolated product, andwherein the ratio of the product to at least one impurity is increasedcompared to a corresponding method in which no detectable amount ofarginine derivative is used in a wash solution.
 20. The method of claim1, wherein the eluate comprises an isolated product, and wherein theratio of the product to host cell protein is increased compared to acorresponding method in which less than about 0.1 M of argininederivative is used in a wash solution.
 21. The method of claim 1,wherein the eluate comprises a product, and wherein the ratio of theproduct to host cell protein is increased compared to a correspondingmethod in which no detectable amount of arginine derivative is used in awash solution.
 22. The method of claim 1, wherein the turbidity of theeluate is reduced compared to a corresponding method in which less thanabout 0.1 M of arginine derivative is used in a wash solution.
 23. Themethod of claim 1, wherein the turbidity of the eluate is reducedcompared to a corresponding method in which no detectable amount ofarginine derivative is used in a wash solution.
 24. The method of claim1, wherein in step (a), at least one impurity is bound to the productand in step (c), contacting the bound medium with one or more washsolutions removes said at least one impurity that is bound to theproduct.
 25. The method of claim 1, wherein the concentration ofarginine derivative in the wash solution is about 1.0 M.
 26. A methodfor isolating an antibody, the method comprising: (a) providing a loadfluid comprising the antibody and one or more impurities, wherein theantibody is an Fc-containing monoclonal antibody; (b) contacting theload fluid with a Protein A medium, wherein the medium can bind theantibody under conditions suitable for binding the antibody, therebyproviding a bound medium; (c) contacting the bound medium with one ormore wash solutions, wherein at least one wash solution comprises anarginine derivative in a concentration of about 0.1 M to about 2.0 M,thereby providing a washed medium, wherein the arginine derivative isacetyl arginine, agmatine, arginic acid, N-alpha-butyroyl-L-arginine orN-alpha-pyvaloyl arginine; (d) contacting the washed medium with anelution solution under conditions suitable for eluting the antibody; and(e) collecting an eluate comprising the antibody, wherein the ratio ofthe antibody to host cell protein in the eluate is increased and whereinthe eluate has reduced turbidity compared to an eluate recovered in acorresponding method in which no detectable amount of argininederivative is used in a wash solution.
 27. The method of claim 26,wherein in step (c) the pH of the wash solution is about 4.5 to about8.0.
 28. The method of claim 26, wherein the concentration of argininederivative in the wash solution is about 0.1 M to about 0.9 M.
 29. Themethod of claim 28, wherein the pH of the wash solution is about 4.5 toabout 8.0.
 30. The method of claim 28, wherein the method does notcomprise anionic upstream adsorptive filtration.
 31. The method of claim26, wherein the concentration of arginine derivative in the washsolution is about 1.1 M to about 2.0 M.
 32. The method of claim 26,wherein the concentration of arginine derivative in the wash solution isabout 0.5 M.
 33. The method of claim 6, wherein the concentration ofarginine derivative in the wash solution is about 1.0 M.
 34. A methodfor reducing turbidity in an eluate comprising a product, the methodcomprising: (a) providing a load fluid comprising the product and one ormore impurities, wherein the product is an Fc-containing monoclonalantibody; (b) contacting the load fluid with a medium, wherein themedium is a Protein A chromatography column, and wherein the medium canbind the product under conditions suitable for binding the product,thereby providing a bound medium; (c) contacting the bound medium withone or more wash solutions, wherein at least one wash solution comprisesan arginine derivative, thereby providing a washed medium, wherein thearginine derivative is acetyl arginine, agmatine, arginic acid,N-alpha-butyroyl-L-arginine or N-alpha-pyvaloyl arginine; (d) contactingthe washed medium with an elution solution under conditions suitable foreluting the product, thereby generating an eluate comprising theproduct; and (e) adjusting the pH of the eluate to between about 6.5 andabout 8.2, wherein the eluate has reduced turbidity compared to acorresponding method in which less than about 0.1M of argininederivative is used in a wash solution.
 35. The method of claim 34,wherein in step (c) the wash solution comprises an arginine derivativein a concentration of about 0.1 M to about 2.0 M.
 36. The method ofclaim 34, wherein in step (c) the wash solution comprises an argininederivative in a concentration of about 0.1 M to about 0.9 M.
 37. Themethod of claim 34, wherein in step (c) the wash solution comprises anarginine derivative in a concentration of about 1.1 M to about 2.0 M.38. The method of claim 34, wherein in step (c) the wash solutioncomprises an arginine derivative in a concentration of about 0.5 M. 39.The method of claim 34, wherein the pH of the wash solution is about 4.5to about 8.0.
 40. The method of claim 34, wherein the method does notcomprise anionic upstream adsorptive filtration.
 41. The method of claim34, wherein the concentration of arginine derivative in the washsolution is about 1.0 M.
 42. A method for reducing turbidity andimpurities in an eluate comprising a product, the method comprising: (a)providing a load fluid comprising the product and one or moreimpurities, wherein the product is an Fc-containing monoclonal antibody;(b) contacting the load fluid with a medium, wherein the medium is aProtein A chromatography column, and wherein the medium can bind theproduct under conditions suitable for binding the product, therebyproviding a bound medium; (c) contacting the bound medium with one ormore wash solutions, wherein at least one wash solution comprises anarginine derivative, thereby providing a washed medium, wherein thearginine derivative is acetyl arginine, agmatine, arginic acid,N-alpha-butyroyl-L-arginine or N-alpha-pyvaloyl arginine; (d) contactingthe washed medium with an elution solution under conditions suitable foreluting the product, thereby generating an eluate comprising theproduct; and (e) adjusting the pH of the eluate to between about 6.5 andabout 8.2, wherein the ratio of the product to at least one impurity inthe eluate is increased and the eluate has reduced turbidity compared toa corresponding method in which less than about 0.1M arginine derivativeis used in a wash solution.
 43. The method of claim 42, wherein in step(c) the concentration of the arginine derivative in the wash solution isabout 0.1 M to about 2.0 M.
 44. The method of claim 42, wherein in step(c) the concentration of the arginine derivative in the wash solution isabout 0.1 M to about 0.9 M.
 45. The method of claim 42, wherein in step(c) the concentration of the arginine derivative in the wash solution isabout 1.1 M to about 2.0 M.
 46. The method of claim 42, wherein in step(c) the concentration of the arginine derivative in the wash solution isabout 0.5 M.
 47. The method of claim 42, wherein the concentration ofarginine derivative in the wash solution is about 1.0 M.